Cochlear degeneration is a major cause of sensorineural hearing loss (SNHL), and the lack of spontaneous regeneration contributes to the irreversible nature of SNHL. Prior studies have examined the utility of forced differentiation of hair cells in the degenerating cochlea, yet it remains unclear whether a restoration in cell number via cell proliferation can also aid cochlear regeneration. Recent work demonstrates that activation of canonical Wnt signaling via genetic or pharmacologic manipulation induces proliferation in the neonatal cochlea. Also, the competence to proliferate in response to Wnt signals is observed in both cochlear supporting cells and tympanic border cells below the basilar membrane. The goal of this proposal is to investigate whether supplementation of Wnt signals can stimulate proliferation and/or regeneration after hair cell degeneration in both the neonatal and mature cochleae. We will initiate degeneration with aminoglycoside application or via a transgenic strategy and concurrently fate-map supporting cells and tympanic border cells in vitro and in vivo. In parallel experiments using multiple transgenic mouse strains, we will study ablation targeting sensory hair cells or supporting cell subtypes. Wnt activation is achieved by using the Cre-Lox system in transgenic mice or local application of Wnt agonists and whether they will initiate proliferation of and regeneration by supporting cells and tympanic border cells are examined. The degree of damage and possible recovery in the cochlea are examined histologically and correlated with pre- and post-treatment auditory physiology in the whole animal. To gain an unbiased insight into the genetic signature of the damaged cochlea, supporting cells and tympanic border cells from undamaged and damaged cochlea are isolated via flow cytometry and subjected to gene array analyses. All transgenic mouse strains, pharmacologic agents, expertise to manipulate and examine the cochlea in vitro and in vivo, and techniques to isolate and enrich cochlear cells are at hand. Together, our research will determine 1) whether Wnt supplementation can help initiate cochlear regeneration and 2) additional targets to enhance this regenerative process in both the neonatal and mature cochleae.